The object of this study was to examine a connected series of phenotypes on a pathway to alcoholism, and to test for allelic associations with multiple polymorphisms across alcohol dehydrogenases (ADH1A, ADH1B, ADH1C, ADH4, ADH5, ADH6 and ADH7) and the high-affinity aldehyde dehydrogenase (ALDH2). Although there have been many genetic association studies on these genes, they have either concentrated on a single phenotype (usually AD), or on a narrow range of genetic markers, or both. There have been suggestions that the polymorphisms studied have not been causative, or that they account for only part of the effects located within these genes; there have been questions about the applicability of results across populations; and a greater integration of information about gene and enzyme variation, the subjective effects of alcohol, habits of alcohol use and symptoms of dependence have been needed. We have found that the effects of the much-studied ADH1B Arg48His polymorphism can be traced through these alcohol-related phenotypes, and that the most easily detected effects are on the phenotypes near the start of the proposed sequence of events. On the whole, our results confirm the prevailing theory, but with some additions and with some features which require further consideration.
None of the ALDH2
SNP-phenotype associations reached the study-wide level of significance of 2.3 × 10−4
. However, it would be unwise to dismiss the possibility of a true effect of variation in ALDH2
in Europeans; precautions against false-positive results can lead to false negatives even with large sample size. A number of phenotypes related to AD, particularly the symptom score for dependence, were associated with rs2238151 at P
≈ 0.001 and with rs737280 at P
< 0.01 (Table ). In a previous study, on a smaller group who had participated in our Alcohol Challenge Twin Study, we found that these two SNPs were associated with significant differences in alcohol metabolism (22
). We postulated that this could be due to a variant which decreased the activity of ALDH2, to a lesser extent than the Asian rs671 polymorphism, and predicted that people homozygous for C at rs2238151 and/or for G at rs737280 should have a lower prevalence of AD and a lower symptom score. That was not shown among the 376 people typed for ALDH2
SNPs in our previous study, but with the larger number of subjects typed in this study we note that the T allele at rs2238151 showed higher AD symptom scores, with P
-values around 0.001 and in the predicted direction. Further studies or meta-analysis on ALDH2
variants in Europeans seem justified. However, neither of these two SNPs showed associations with self-reported flushing or with alcohol consumption so the mechanism of any effect on dependence is unclear.
Our main finding is that the variation at rs1229984, the well-known ADH1B Arg48His
polymorphism, is associated with flushing or other reactions to alcohol as well as with alcohol consumption in Europeans. Self-reported unpleasant reactions to alcohol are known to occur in a substantial proportion of people, particularly women [see Table , and (56
)], but our earlier attempts to test whether ADH1B
variation affected these reactions (57
) were inconclusive. With larger numbers in the present study, the ADH1B 48His
allele, which has a frequency of around 5% in this group, is highly significantly associated with more frequent alcohol reactions and lower alcohol intake. The effect of this polymorphism on reported alcohol intake is summarized in Figure and Table ; for both men and women, the median alcohol intake was notably lower in heterozygotes than in 48Arg
homozygotes, and in the small number of 48His
homozygotes alcohol consumption was very low.
However, this polymorphism is not significantly associated, at least at our study-wide significance level, with AD or AD symptom count. Practically, all previous studies, in Asia and in Europe, have found that the 48His
allele is protective against AD. For Europeans, the relative risk of AD for heterozygotes against 48Arg
homozygotes has been estimated at 0.47 with 95% confidence intervals of 0.29 to 0.76 (36
). Our estimated relative risk for DSM-IIIR AD in heterozygotes compared to 48Arg homozygotes, from the current data, is 0.57 for men and 0.49 for women. There is no evidence of heterogeneity and the pooled odds ratio estimate is 0.53 with 95% confidence intervals 0.32–0.88. The point to note is that our results are compatible with the expected size of the effect on AD risk. As noted above in relation to ALDH2
variation, failure to meet a stringent significance level does not exclude an effect; and the AD symptom score phenotype showed association with rs1229984 at the P
< 0.002 level. On balance, we consider that rs1229984 does affect AD risk but the nature of our study (with subjects drawn from the general population), the low MAF for this polymorphism, and the stringent study-wide significance level arising from the examination of multiple SNPs and phenotypes, contribute to our inability to demonstrate this unequivocally.
A number of previous reports have considered whether variation at ADH1B Arg48His
affects the alcohol flush reaction or alcohol reactions in general, both in Asians (38
) and in Europeans. The results are very mixed; in Europeans a measure of ‘level of response’ to alcohol was affected by this polymorphism (59
) but alcohol-induced flushing was not (60
). In Asians also there are about equal numbers of positive and negative reports, and some suggestion of gene–gene interaction in that ADH1B
genotype may only affect flushing in ALDH2
heterozygotes. However, our results on this point are strong, and we conclude that ADH1B Arg48His
does affect the immediate response to alcohol consumption.
No other polymorphisms in either ADH or ALDH2 had significant or suggestive associations with alcohol reactions. However, many ADH SNPs showed associations with drinking behavior (Fig. and Table ), some because of LD with rs1229984. When the effect of rs1229984 was regressed out, there was an independent association between rs1042026 in ADH1B and the overall quantity phenotype (P = 4.7 × 10−5), with the A-allele conferring higher alcohol intake on average over the previous 12 months. Smaller associations between rs1230165 in ADH5 and frequency of alcohol use, and between both rs37262894 (in ADH4) and rs1693482 (ADH1C Arg272Gln) and the maximum number of drinks consumed in 1 day in the previous 12 months were also observed. No SNPs producing stronger effects than rs1229984, or explaining the effects at rs1229984 through LD with a distinct causative variant, were found. However, there were indications of independent effects in ADH1B, ADH1C, ADH4 and ADH5 which did not reach our required significance level, but which still need to be confirmed or refuted.
Comparable data are available from three other studies with SNP coverage of the ADH
region and one of ADH4
only, which focused on associations with AD. Using a conventional test for allelic association, Luo et al
) found significant results for rs1229984 (Arg48His
) in European-Americans and for rs2066702 (Arg369Cys)
in African-Americans, both in ADH1B
. Using an analysis based on deviation from HWE, there were indications that variation in ADH1A, ADH1B, ADH4, ADH5
affected dependence risk (20
). Significant associations between AD in European-Americans and SNPs in ADH4, ADH1A
were found in another ADH
-cluster-wide study by the Collaborative Study on the Genetics of Alcoholism (19
). While no association was detected between rs1229984 and AD in European-Americans, in African-Americans rs2066702 was significantly (P
< 0.05) associated with AD. The most significant associations were in the ADH1A-ADH1B
region or in ADH4
, depending on the AD criteria used. It is interesting to note that out of 110 SNPs tested on 2139 people, three were significant at the 0.01 level and a further 18 had P
-values between 0.05 and 0.01; we can infer that the effects on AD risk are either small or they are associated (as we found) with polymorphisms with low minor allele frequencies. A further study with recruitment in Ireland (21
) found associations at P
< 0.01 between AD and SNPs in ADH1B
, and at P
< 0.05 in ADH1A, ADH1B, ADH1B
. Significant association between AD and ADH4
SNPs was also reported in Brazilian subjects (61
). Overall, the genes coding for Class I ADHs with high affinity for ethanol are the ones which most frequently show significant effects on dependence risk, but ADH4
has also been implicated in several studies.
From our results and the published literature, we can now propose that rs1229984, or ADH1B Arg48His
, affects the subjective experiences associated with alcohol use, specifically through flushing or similar reactions, even in Europeans homozygous for the active form of ALDH2
. This affects alcohol use, which in turn affects AD risk. Other polymorphisms in ADH
genes may also affect AD through similar mechanisms, but no strong evidence of this is yet available. The main anomaly in this narrative is that rs1229984 probably does not affect the rate of alcohol metabolism, at least so far as it can be measured from changes in blood and breath alcohol concentrations (51
). It still remains possible that this variant affects the steady-state concentration of acetaldehyde within the hepatocytes, which is where acetaldehyde is produced, and that this affects the release of vasoactive compounds into the circulation and causes alcohol-induced flushing. Measurement of acetaldehyde concentrations in the liver of humans seems beyond our reach, but measurements of histamine or other vasoactive molecules in the circulation might allow testing of this concept; and transgenic mice expressing rs1229984 might allow more invasive studies of the effects of this polymorphism on alcohol and acetaldehyde metabolism.